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Soluble methane monooxygenase (sMMO) catalyzes the oxidation of methane to methanol. Single-turnover reactions of sMMO from Methylococcus capsulatus (Bath) were studied by stopped-flow optical spectroscopy to examine further the activated dioxygen intermediates and their reactions with hydrocarbon substrates. A diiron(III) peroxo species designated Hperoxo is the first intermediate observed in the reaction between the chemically reduced hydroxylase (Hred) and dioxygen. The optical spectrum of this species determined by diode array detection is presented for the first time and exhibits visible absorption bands with λmax ≈ 420 nm (ε = 4000 M-1 cm-1) and λmax = 725 nm (ε = 1800 M-1 cm-1). The temperature dependences of the rate constants for formation and decay of Hperoxo and for the subsequent intermediate, Q, were examined in the absence and in the presence of hydrocarbon substrates, and activation parameters for these reactions were determined. For single-turnover reaction kinetics monitored at 420 nm, the λmax for Q, a nonlinear Eyring plot was obtained when acetylene or methane was present in sufficiently high concentration. This behavior reflects a two-step mechanism, Q formation followed by Q decay, in which the rate-determining step changes depending on the temperature. The rate of Hperoxo formation does not depend on dioxygen concentration, indicating that an effectively irreversible step involving dioxygen precedes formation of the diiron(III) peroxo species. The rate constant observed at 4 °C for Hperoxo formation, 1−2 s-1, is slower than that determined previously by Mössbauer and optical spectroscopy, ∼20−25 s-1 (Liu, K. E.; et al. J. Am. Chem. Soc. 1995, 117, 4997−4998; 10174−10185). Possible explanations for this discrepancy include the existence of two distinct peroxo species. Intermediate Q exhibits photosensitivity when monitored by diode array methodology, a property that may arise from enhanced reactivity of a transient charge-transfer species. The photoreaction can be avoided by using a monochromator to obtain kinetics data at single wavelengths. The reactions of substrates with intermediate species were studied by single- and double-mixing stopped-flow spectroscopy. The Q decay rate exhibits an approximate first-order dependence on substrate concentration for a wide range of hydrocarbons, the relative reactivity varying according to the sequence acetylene > ethylene > ethane > methane > propylene > propane. In addition, the data indicate that Hperoxo can oxidize olefins but not acetylene or saturated hydrocarbons.

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The elaborate process of genomic replication requires a large collection of proteins properly assembled at a DNA replication fork. Several decades of research on the bacterium Escherichia coli and its bacteriophages T4 and T7 have defined the roles of many proteins central to DNA replication. These three different prokaryotic replication systems use the same fundamental components for synthesis at a moving DNA replication fork even though the number and nature of some individual proteins are different and many lack extensive sequence homology. The components of the replication complex can be grouped into functional categories as follows: DNA polymerase, helix destabilizing protein, polymerase accessory factors, and primosome (DNA helicase and DNA primase activities). The replication of DNA derives from a multistep enzymatic pathway that features the assembly of accessory factors and polymerases into a functional holoenzyme; the separation of the double-stranded template DNA by helicase activity and its coupling to the primase synthesis of RNA primers to initiate Okazaki fragment synthesis; and the continuous and discontinuous synthesis of the leading and lagging daughter strands by the polymerases. This review summarizes and compares and contrasts for these three systems the types, timing, and mechanism of reactions and of protein-protein interactions required to initiate, control, and coordinate the synthesis of the leading and lagging strands at a DNA replication fork and comments on their generality.

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